Agents for blocking T cell mediated immune reactions

ABSTRACT

Agents for blocking T cell-mediated immune reactions are provided. Such agents are peptides, referred to hereinafter as “CD28 peptide mimetics”, of from 15 to 30 amino acids in length. The CD28 peptide mimetics comprise the hexapeptide motif ‘MYPPPY’ or a retro-inverso isomer thereof. The CD 28 peptide mimetics further comprise flanking sequence at the amino and carboxyl terminus of the hexapeptide motif. Such flanking sequence permit the CD28 peptide mimetic to assume a polyproline II (PPII) conformation when placed in water or a buffered solution at physiological pH and a temperature of about 25° C. Methods for treating subjects with T cell mediated autoimmune diseases or disorders are also provided Such methods comprise administering one or more of the CD 28 peptide mimetics to a subject which has such disease or disorder 30. The present invention also relates to a method of blocking activation and proliferation of CD4+ cells. The method comprises contacting such cells with one or more of the present CD 28 peptide mimetics.

[0001] This application claims priority to U.S. Provisional Application60/252,744 filed November Nov. 22, 2000 and U.S. Provisional Application60/250,984 filed Dec. 4, 2000, both of which are incorporated herein intheir entirety.

BACKGROUND

[0002] The present invention relates to agents and methods for blockingdeleterious T cell mediated immune reactions. Such reactions occur inautoimmune diseases, such as for example, multiple sclerosis (MS),rheumatoid arthritis, systemic lupus erythematosis, psoriasis, diabetes,and allergies. Such reactions also occur during rejection oftransplants.

[0003] Two types of signals are required for T cell activation andproliferation. The first, which gives specificity to the immuneresponse, involves an interaction between the T-cell receptor/CD3complex and an antigenic peptide presented by major histocompatibilitycomplex (MHC) class I or class II proteins on the surface of anantigen-presenting cell (APC). The second type of signal, called acostimulatory signal, involves interaction between receptor-ligand pairsexpressed on the surface of APCs and T cells. Antigenic stimulation inthe absence of costimulation mechanisms induces a state ofunresponsiveness or anergy and eventual cell death by apoptosis in theresponding T cells. Thus, antigenic stimulation in the presence ofcostimulation prevents anergy and cell death, thereby promoting cellsurvival.

[0004] CD 28 is one of the principalT cell costimulatory receptors. CD28binds APC costimulatory ligands B7.1(CD80) and B7.2(CD86). CD28 is atransmembrane homodimer that is constitutively expressed on 90% ofmammalian CD4+ T cells. Upon binding to the B7 family of molecules, CD28delivers a powerful costimulatory signal for T cell activation andclonal expansion. Engagement of CD28 by its ligands B7-1 or B7-2 on thesurface of APCs initiates a signaling cascade culminating in cytokineproduction and expansion of specific T-cells.

[0005] T cells also express cytotoxic-T-lymphocyte antigen 4, CTLA-4(CD152), a close relative of CD28. In contrast to CD28, CTLA-4 is notexpressed by naive T cells but is rapidly induced after T cellactivation. Analogous to CD28, CTLA-4 also binds B-7 family ofmolecules, albeit with higher avidity than CD28. Engagement of CTL4 withthe B7 ligands transmits a negative signal to the T cells, thusterminating the immune response.

[0006] The critical role played by the B7/CD28:CTLA-4 costimulatoryinteraction in determining the fate of immune responses (activation vsanergy/apoptosis) makes it an attractive target for therapeuticimmunomodulation in a wide range of autoimmune diseases. Recently, ithas been shown that administration of monoclonal antibodies to B7molecules and CTLA-4 Ig fusion protein ameliorate autoimmune diseases invarious animal models, including (EAE) an animal model for MS, diabetesand systemic lupus erythematosis. However, these results depend upon thetiming of antibody administration. For example, CTLA-4 Ig was noteffective in blocking already established EAE, and anti B7-2 mAB andanti CTLA-4 mAB exacerbate the disease. Moreover, the value of suchantibodies as effective therapeutic agents is limited by virture oftheir inherent immunogenicity and poor penetration across tissuebarriers

[0007] Accordingly, it is desirable to have additional methods andagents which can be used to block deleterious T-cell mediated immunereaction and to ameliorate the symptoms of diseases and disordersassociated with such reactions.

SUMMARY OF THE INVENTION

[0008] The present invention provides new agents for blocking Tcell-mediated immune reactions. Such agents are peptides, referred tohereinafter as “CD28 peptide mimetics”, of from 15 to 30 amino acids inlength. The CD28 peptide mimetics comprise the hexapeptide motif‘MYPPPY’ or a retro-inverso isomer thereof. The CD 28 peptide mimeticsfurther comprise flanking sequence at the amino and carboxyl terminus ofthe hexapeptide motif. Such flanking sequence permit the CD28 peptidemimetic to assume a polyproline II (PPII) conformation when placed inwater or a buffered solution at physiological pH and a temperature ofabout 25° C.

[0009] The present invention relates to methods for treating subjectswith T cell mediated autoimmune diseases or disorders. Such methodscomprise administering one or more of the CD 28 peptide mimetics to asubject which has such disease or disorder. 30.

[0010] The present invention also relates to a method of blockingactivation and proliferation of CD4+ cells. The method comprisescontacting such cells with one or more of the present CD 28 peptidemimetics.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1: (A) CD spectra of end group blocked L CD28 (ELCD28) andretro-inverso CD28 peptide (RICD28) at 400 μM in PBS, 50% TFE at 25° C.and, (B) at 35° C. Mean residue ellipticity (θ) is expressed in degreescm².dmol⁻¹.

[0012]FIG. 2: Affinity and kinetics of EL CD28 peptide binding toCD80-Ig. (A) A range of EL CD28 peptide concentrations (375 nM to 18 μM)were injected for 300 s at 10 μl/min through a flow cell with CD80-Ig(1488 RU) or no protein (control) immobilized. (B) Injection phaseanalysis of a representative binding curve and the residual followinginjection of EL CD28 peptide (6 μM) over CD80-Ig immobilized and controlflow cell. (C) A plot of (dR/dt) against RU was calculated from thesensograms and the slope of this plot was plotted against concentrationof ELCD28 peptide. The association rate constant of the interactionbetween EL CD28 and CD80-Ig, as measured from the slope of this plot, isindicated. The dissociation rate constant is obtained from theintercept. The data yields a K_(d) of 2.34 μM for the interactionbetween EL CD28 peptide and CD80-Ig.

[0013]FIG. 3: Affinity and kinetics of RI CD28 peptide binding toCD80-Ig. (A) A range of RICD28 peptide concentrations (375 nM to 18 μM)were injected for 300 s at 10 μl/min through a flow cell with CD80-Ig(1512 RU) or no protein (control) immobilized. (B) Injection phaseanalysis of a representative binding curve and the residual, followinginjection of RI CD28 peptide (6 μM) over CD80-Ig i m mobilized andcontrol flow cell. (C) A plot of (dR/dt) against RU was calculated fromthe sensograms and the slope of this plot was plotted against theconcentration of RICD28 peptide. The association rate constant of theinteraction between RI CD28 and CD80-Ig, as measured from the slope ofthis plot, is indicated. The dissociation rate constant is obtained fromthe intercept. The data yields a K_(d) of 2.53 μM for the interactionbetween EL CD28 peptide and CD80-Ig.

[0014]FIG. 4: Competitive kinetics between CD28 APR and CD28-Ig forbinding immobilized CD80-Ig. (A) An overlay of sensograms obtained frominjection of a mixture of CD28-Ig at constant concentration (3.2 μM) andEL CD28 peptide at varying concentration (375 μM to 6 μM) at 10 μl/minover a flow cell with bound CD80-Ig (322 RU). The top curve representsthe binding of CD28-Ig alone in the absence of competing peptide. Theresponse of CD28-Ig binding decreases with increasing concentration ofEL CD28 peptide. (B) An overlay of sensograms obtained from injection ofa mixture of CD28-Ig at constant concentration (3.2 μM), and RI CD28peptide at varying concentration (375 nM to 6 μM) at 10 μl/min over aflow cell with bound CD80-Ig (338.6 RU). The top curve represents thebinding of CD28-Ig alone in the absence of competing peptide.

[0015]FIG. 5. Antigen-specific T-cell proliferative responses of lymphnode cells and splenocytes from MBP peptide-specific TCR transgenic micethat carry a Vα4 Vβ 8.2 TCR treated with CD28 peptides. Single cellsuspensions of CD4⁺ T cells isolated from the (A) lymph nodes and (B)spleen (5×10⁴ cells /well) were stimulated with the encephalitogenicpeptide of MBP, NAc 1-11 (10 μg/ml) and cultured for a total of 72 hr(including an 18 hr pulse with H³ thymidine) in the presence of varyingconcentrations of CD28 peptide analogues as shown. Data represents meanthymidine uptake and is plotted as delta cpm±SE. Results are mean ofthree different experiments. Proliferative responses of CD4+ LNC andspleen cells treated with L CD28, end group blocked CD28 andretro-inverso CD28 at all concentrations used were significantly lessthan untreated and control peptide (Reverse L CD 28 and D CD 28) treatedcells *=p<0.01 by ANOVA.

[0016]FIG. 6: Fewer cells secrete IL-2 when stimulated in the presenceof CD28 APR. CD4+ T cells were isolated from the pooled lymph nodes ofVβ8.2 Vα 4 TCR transgenic mice and stimulated with 10 μg/ml of NAc 1-11peptide of MBP either alone or in the presence of the specifiedconcentrations CD28 APR and assayed by ELISASPOT. Significant decreasein the number of NAc1-11 responders was observed following treatmentwith EL CD28 and RI CD28 @ 75 μM and 100 μM concentrations. Results arerepresentative of two experiments *=p<0.05 by one way ANOVA

[0017]FIG. 7: (A) CD28 synthetic peptide treatment increases apoptosisof CD4+ T cells in vitro. 5×10⁵ CD4+ T cells were isolated from thepooled lymph nodes of Vβ8.2 Vα 4 TCR transgenic mice and stimulated with10 μg/ml of NAc 1-11 peptide of MBP either alone or in the presence ofthe specified concentrations CD28 APR. Lymph node cells were harvestedafter 48 hrs and apoptotic cells among Vβ8.2+ cells were detected by theTUNEL methods and analyzed by flow cytometry. The data shown is theaverage results from three experiments. Significant increase in thepercentage of apoptotic cells was observed following treatment with 150μM EL CD28 and 75 μM or 150 μM RI CD28 APR. Lymph node cells alone orwhen treated with the CD28 peptides in the absence of antigen showedmaximum apoptosis (Data not shown). *=p<0.01 by one way ANOVA.Representative histograms showing increase in FITC conjugated TUNELpositive cells in cultures treated with 120 μM EL CD28 or RI CD28 areshown in (B) and (C) respectively

[0018]FIG. 8. Injection of synthetic CD28 peptide analogues inhibitsdevelopment of clinical EAE and attenuates established EAE.

[0019] B10.PL mice were immunized with GP-MBP in CFA and injectedpertussis toxin i.p on day 0 and 2. The data are presented as the meanclinical score in each group on different days of observation. Animalsreceived a single i.v. injection of 500 g of ELCD28(n=10) or RICD28(n=10) peptides or control [LCD28(n=12), RLCD28(n=6) and D CD28(n=6)] peptides or PBS (n=12) on the day of immunization (A) or on day14 post-immunization (B).Data represent the means of pooled data fromtwo separate experiments.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides peptides, i.e. CD28 peptidemimetics and compositions containing such peptides, which are useful forblocking or inhibiting activation and proliferation of T cells,particularly CD4+ cells.

[0021] The CD 28 peptide mimetics are from 15 to 30 amino acids,preferably from 17 to 25 amino acids, more preferably from 19 to 21amino acids in length. The CD28 peptide mimetics comprise a hexapeptidewith the following sequence: MYPPPY, SEQ ID NO. 1, or the retro-inversoisomer thereof, i.e., YPPPYM, SEQ ID NO. 2. The CD28 peptide mimeticsfurther comprise flanking sequences, i.e. a plurality of amino acids, atthe amino and a plurality of amino acids at carboxy termini of thehexapeptide motif. Preferably, the hexapeptide is sandwiched between twoampiphilic, anti-parallel right-twisted B strands. For optimum stabilityof the CD28 peptide mimeic, it is preferred that the antiparallel Bstrands be of the same length.

[0022] In those instances where the methionine is at the amino terminusof the hexapeptide, the CD 28 peptide mimetic, referred to hereinafteras “L” peptide mimetic, is comprised of levorotary amino acids. In thoseinstances where the methionine is at the carboxy terminus of thehexapeptide, the CD 28 peptide mimetic, referred to hereinafter as a “D”peptide mimetic, is comprised of dexorotary amino acids such that the Dpeptide mimetic is a topochemical equivalent of the corresponding Lpeptide mimetic. The retro-inverso modification of the L-peptide mimeticto produce a corresponding D peptide mimetic involves the reversal ofall amide bonds within the peptide backbone. This is achieved byreversing the direction of sequence and inverting the chirality of eachamino acid residue by using D-amino acids. The goal of this topochemicalapproach is to create an analog such that the reversed amide bonds inthe D peptide mimetic retains both the planarity and conformationalrestrictions of peptide bonds (CONH) and the spatial orientation of sidechains remains closely related to that of the corresponding L peptidemimetic. Advantageously, the D peptide mimetic is resistant to proteasesthat are present in mammals.

[0023] The amino and carboxy termini of the CD 28 peptide mimetics maybe free or, preferably, end-blocked. When placed in water or a bufferedsolution having a pH of about 7.4, the CD 28 peptide mimetics adopt aPPII helical conformation. The secondary structure of the CD 28 peptideand the presence of a PPII helical conformation may be determined usinga circular dichromism assay.

[0024] The CD 28 peptide mimetics bind B7 molecules on APC's (AntigenPresenting Cells) with an affinity which, preferably is less than theaffinity of CTLA-4 for these molecules. The CD 28 peptide mimetics bindB7 molecules with an affinity which is equivalent to the affinity ofCD28 for these ligands. This property can be determined empiricallyusing a competive binding analaysis. Alternatively, the relativeaffinity of the CD 28 peptide mimetic for B7 molecules can be estimatedon the basis of Ka and Kd measurements.

[0025] The CD 28 peptide mimetic has a Kd which is equivalent to the Kdof CD28. The CD 28 peptide mimetic has a Kd, preferably, between 2 and 3micromoles, more preferably, between 2.1 and 2.7 micromoles. Thus, theCD 28 peptide mimetic binds to the B71 ligand and the B7 2 ligand, whichare also known as CD 80 and CD 86 respectively, with fast kinetics.

[0026] In certain embodiments, the L form of the CD28 peptide mimeticcomprises the following sequence: FMYPPPYL, SEQ ID NO 3 Thecorresponding D form of this peptide mimetic is the retro inverso isomerof this sequence. In certain embodiments, the D form of the CD 28peptide mimetic comprises the sequence LYPPPYMFEIK, SEQ ID NO. 4. In oneembodiment, the CD28 peptide mimetic is an L-peptide which comprises 20L-amino acids, has the sequence KIEFMYPPPYLDNERSNGIE, SEQ ID NO. 5, andhas free ends. In another embodiment, the peptide mimetic is anL-peptide which comprises 20 L-amino acids, has the sequenceKIEFMYPPPYLDNERSNGIE, SEQ ID NO. 5 and has blocked ends; i.e., thelysine at the amino terminus is acetylated and the glutamic acid at thecarboxy terminus is amidated. In a further embodiment, the peptidemimetic is a D-peptide which comprises 20 D-amino acids and has thesequence EIGNSRENDLYPPPYMFEEK, SEQ ID NO. 6, and has free ends. Inanother embodiment, the peptide mimetic is a D-peptide which comprises20 D-amino acids and has the sequence EIGNSRENDLYPPPYMFIEK, SEQ ID NO.6, wherein the aspartic acid residue at the amino terminus is acetylatedand the lysine residue at the carboxy terminus is amidated. In certainembodiments of the L form of the CD28 peptide mimetic, flanking regionsof the core hexapeptide comprise a repetitive LS sequence; while theflanking regions of the corresponding D form of the CD28 peptide mimeticcomprise a repetitive SL sequence. Thus, the CD 28 peptide mimetic maycomprise one of the following sequences

[0027] LSLSLSMYPPPYLSLSLS, SEQ ID NO. 7,

[0028] LSLSLSKEIFMYPPPYLDNESLSLSLS, SEQ ID NO. 8,

[0029] SLSLS1lYPPPYMSLSLSL SEQ ID NO. 9, and

[0030] SLSLSLENDLYPPYMFIEKSLSLSL, SEQ, ID NO. 10.

[0031] The present CD 28 peptide mimetics also encompass peptides thatare biologically equivalent variants of SEQ ID NO. 5, SEQ ID NO. 6, SEQID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. 10 . A“biologically equivalent variant” as used herein, refers to a peptidewhose amino acid sequence is similar but not identical to the amino acidsequence of one of these sequences, hereinafter referred to as the“reference” amino acid sequence, but does not have 100% identity withsuch reference sequence. Peptides which are biologically equivalentvariants have an altered sequence in which one or more of the aminoacids in the reference sequence other than the hexapeptide motif, i.e.,in the flanking sequence, is substituted, or in which one or more aminoacids are deleted from or added to one or both of the flanking sequencesof the hexapeptide motif. Preferably the deletions and additions arelocated at the amino terminus, the carboxy terminus, or both, of one ofthe sequences shown above.

[0032] While it is possible to have nonconservative amino acidsubstitutions, it is preferred that the substitutions be conservativeamino acid substitutions, in which the substituted amino acid hassimilar structural or chemical properties with the corresponding aminoacid in the reference sequence. By way of example, conservative aminoacid substitutions involve substitution of one aliphatic or hydrophobicamino acid, e.g. alanine, valine, leucine and isoleucine, with another;substitution of one hydroxyl-containing amino acid, e.g. serine andthreonine, with another; substitution of one acidic residue, e.g.glutamic acid or aspartic acid, with another; replacement of oneamide-containing residue, e.g. asparagine and glutamine, with another;replacement of one aromatic residue, e.g. phenylalanine and tyrosine,with another; replacement of one basic residue, e.g. lysine, arginineand histidine, with another; and replacement of one small amino acid,e.g., alanine, serine, threonine, methionine, and glycine, with another.As a result of the alterations, such the biologically equivalent varianthas flanking amino acid sequences which are at least 70% identical,preferably at least 80% identical, more preferably at least 90%identical to the flanking amino acid sequence of reference sequence.Variant sequences, which are at least 90% identical, have no more than 1alteration, i.e., any combination of deletions, additions orsubstitutions, per 10 amino acids of the flanking amino acid sequence.Percent identity is determined by comparing the amino acid sequence ofthe variant with the reference sequence using MEGALIGN module in the DNASTAR program.

[0033] Peptides which are biologically equivalent variants of CD 28peptide mimetics comprising SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7,SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. 10 bind to B71 and B72 withan affinity that is less than CTLA-4 and comparable to CD28. Peptideswhich are biologically equivalent variants of CD 28 peptide mimeticscomprising SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, andSEQ ID NO. 9 have a Kd which is between 2 and 3 micromoles.

[0034] The present invention also provides a method of treating thesymptoms of a disease or disorder that involves a deleterious activationof T cells. Examples of such diseases are autoimmune diseases, such asMS, EAE which is the mouse model for MS, rheumatoid arthritis, andinsulin-dependent diabetes mellitus. One example of another disorderthat involves deleterious activation of T cells is rejection of anallograft transplant Such method comprises administering apharmaceutical composition, which comprises an L CD 28 peptide mimetic,a D CD 28 peptide mimetic or both to a subject in need of the same. Asused herein, the term subject refers to a mammalian animal, preferably ahuman. By “treating” is meant ameliorating or tempering the severity ofthe disorder or the symptoms associated therewith. In such cases, as forexample multiple sclerosis, the pharmaceutical composition isadministered either when patients have clinical symptoms, or when agenetic mutation indicative of MS is identified. Preferably, theprotocol involves oral administration of a pill or water-solublemixture, or injection, preferably intravenous injection, of thepharmaceutical composition. In the case of rheumatoid arthritis, thepharmaceutical composition may be administered when patients exhibitclinical symptoms of the disease. In the case of insulin-induceddiabetes mellitis, the pharmaceutical composition is administered whenpatients have clinical symptoms, or when a genetic mutation indicativeof diabetes mellitus is identified. The protocol involves oraladministration of the pharmaceutical composition, which preferably is inthe form of a pill or water soluble mixture, or injection of thepharmaceutical composition, preferably intravenous injection.

[0035] The present invention also relates to a method of blockingactivation and proliferation of CD4+T cells in vitro and in vivo.

[0036] The present invention also relates to a method of preventingT-cell mediated rejection of an allograft transplant. The methodcomprises administering a CD 28 peptide mimetc to a patient that hasrecently undergone, or is about to undergo, such transplant. Preferably,the peptide mimetic is administered to such a patient intravenously.

[0037] Methods of Preparing the Peptide Mimetics

[0038] The CD 28 peptide mimetics are prepared using standard techniquesand equipment for preparing synthetic peptides, such as a synthesizer.For example, the CD 28 peptide mimetics may be prepared using the 9600Millegen/Biosearch synthesizer or a 40 well multiple peptide synthesizer(MPS 396, Advanced Chem Tech, Lousiville, Ky.) and purified by reversephase HPLC (Water's Associates) and characterized by electrosprayionization spectrometry (Mass Spectral facility, OSU). Retro-inversopeptides are assembled in a reverse order of amino acids withFmoc-D-aminoacid derivatives.

[0039] Pharmaceutical Composition

[0040] The pharmaceutical composition comprises a biologically effectiveamount of a CD 28 peptide mimetic, and preferably a relatively inerttopical carrier. Many such carriers are routinely used and can beidentified by reference to pharmaceutical texts.

[0041] The acceptable carrier is a physiologically acceptable diluent oradjuvant. The term physiologically acceptable means a non-toxic materialthat does not interfere with the effectiveness of the antagonist. Thecharacteristics of the carrier will depend on the route ofadministration and particular compound or combination of compounds inthe composition. Preparation of such formulations is within the level ofskill in the art. The composition may further contain other agents whicheither enhance the activity of CD 28 mimetic or complement its activity.The composition may further comprise fillers, salts, buffers,stabilizers, solubilizers, and other materials well known in the art.

[0042] Dosage

[0043] In vivo, a biologically effective amount is an amount sufficientto sufficient to show a meaningful benefit, i.e., partially orcompletely relieve the symptoms associated with the respective diseaseor disorder. The amount of the CD 28 peptide mimetic required willdepend upon the nature and severity of the condition being treated, andon the nature of prior treatments which the subject has undergone andthe type of defect or disease being targeted. Ultimately, the dosagewill be determined using clinical trials. Initially, the clinician willadminister doses that have been derived from animal studies. Aneffective amount can be achieved by one administration of thecomposition. Alternatively, an effective amount is achieved by multipleadministration of the composition to the subject. In vitro, thebiologically effective amount is the amount sufficient to reduceproliferation or activation of CTLA4+ T cells.

[0044] Between 0.125 mg and 5 mg will be administered, in two-foldincrements, to determine the full range of inhibition of EAE andtoxicity of the CD 28 peptide mimetic. The efficacy of oral,subcutaneous and intravenous administration is determined in clinicalstudies.

[0045] Autoimmune Diseases

[0046] Multiple Sclerosis.

[0047] MS is a demyelinating disease of the central nervous system thatlikely results from a combination of genetic susceptibility,environmental factors, pro-inflammatory cytokines released in the CNSand autoimmune reactionsMS pathology consists of CNS inflammatoryinfiltrates containing CD4+ autoreactive T cells and demyelination.Hence, MS is considered to be an autoimmune disease of the CNS that istriggered by an unknown stimulus. A cascade of inflammatory events leadsto the activation of the immune system that perpetuates the inflammatoryprocess. T cells specific for myelin antigens arise as a primary orsecondary event in MS, which damages myelin and destroysoligodendrocytes leading to demyelination. Thus a CD4+ T cell mediatedimmune response plays a central role in the pathogenesis of MS.Depending on the extent of demyelination and rate of remyelination, theMS disease course is variable and is classified as relapsing-remitting,primary progressive, secondary progressive and progressive relapsing.

[0048] The EAE Model:

[0049] EAE is an animal model used to design and test interventions inthe MS disease process because of its clinical, immunological andhistopathological similarities to MS. EAE is an experimental autoimmunedisease of the CNS that results from the immunization of susceptibleanimals with myelin proteins, including myelin basic protein (MBP),proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG) orpeptides derived from these proteins. EAE is mediated by activatedCD4+Tcells that recognize the neuroantigen in the context of majorhistocompatibility complex (MHC) Class II molecules. The resultingimmune response initiates a series of events including CNS mononuclearcell infiltration, demyelination, perivascular edema, and ascendingparalysis. EAE in susceptible mouse strains, including B10.PL, SLJ, andPL/J is characterized by a relapsing remitting (R-EAE) disease course,in which animals undergo an initial acute episode, followed byremission, with progression to multiple relapses. R-EAE is initiated inmice by either immunization with myelin antigens combined with CFA andpertussis toxin (PT) or by adoptive transfer of activated myelinspecificCD4+ T cells. The N acetylated 1-11 (NAc 1-11) peptide of MBP is theimmunodominant epitope recognized by encephalitogenic CD4+ T cells inthe context of MHC Class II I-A^(υ) (H-2^(υ)) in the B10.PL mouse. Inthe immune response to this peptide in I-A^(υ) mice, there ispreferential usage of the Vb8 T cell receptor (TCR) genes. This findinghas led to the development of TCR transgenic mice overexpressing theMBP-specific TCR (Va4 V.

[0050] T Cell Activation and EAE:

[0051] T cell clonal expansion is initiated through the recognition of apeptide MHC complex by the T-cell receptor. Complete T cell activationrequires further signaling via costimulatory molecules. Once activated,naive CD4+ (Th0) cells can develop distinct effector phenotypesdepending on the local cytokine milieu and type of costimulatorymolecules expressed by the antigen presenting cell (APC). IL-12 releasedby APC biases naive cells towards the Th1 phenotype, which ischaracterized by the production of IL-2 and the pro-inflammatorycytokines INF-λ and TNF-α. CD4+T cells of the Th1 phenotype are requiredfor EAE induction in immunocompetent animals. Studies performed ontransgenic animals with EAE have shown that expansion of T cellsspecific for myelin antigens is required to obtain CNS perivascularinflammatory infiltration and demyelination.

[0052] The present invention will be described in greater detail withthe aid of the following examples which should be considered asillustrative and non-limiting.

EXAMPLES Example 1 CD28 Peptide Mimetic Synthesis and Characterization

[0053] Analog design:

[0054] CD28 is a member of a subfamily of molecules within theimmunoglobulin superfamily which contains a single IgV domain. Sequencealignment of the IgV fold revealed a rigorous conservation of ahexapeptide motif “MYPPPY” in the CDR3-like region of CD28. Thelocalization of the motif in the solvent exposed CDR3-like region andconservation across species strongly suggested the presence of acandidate ligand binding epitope in this region. The hydrophobic motifforms a loop that is conformationally constrained due to the presence ofadjacent proline residues.

[0055] Based on molecular modeling of the CD28 extracellular domain, a20 residue linear peptide was defined that comprised the conservedpolyproline motif and flanking sequence such that the predicted sequencehad a greater propensity to form an helical structure as predicted bythe secondary structure algorithm by Chou and Fasman. The sequence ofthis L form of the CD 28 peptide mimetic is KIEFMYPPPYLDNERSNGIE, SEQ IDNO. 5

[0056] In order to mimic the end groups of the ligand binding epitope ofparent CD28 molecule the amino terminus of free L CD28 peptide mimeticwas acetylated and the carboxy terminus was amidated. In addition, thismodification neutralizes charges at the termini of the peptide. Thismodification stabilizes the secondary sturcture, and is expected toenhance the functional interaction of the molecule with B7 ligands.

[0057] Unmodified peptides can be susceptible to enzymatic degradationand rapid clearance from circulation. Accordingly, a retro-inversoisomer of the above described L CD28 peptide mimetic was designed.Retro-inverso peptides are peptides made of reversed D-amino acids, sothey are mirror images of a mirror image. The use of D amino acidsresults in inverted chirality and the reversed order of amide bonds(—NHCO— instead of —CONH—) and creates an analogue that regenerates boththe planarity of peptide bonds and the spatial orientation of sidechains closely related to that of the original peptide. Theretro-inverso peptide was assembled in a reverse order of amino acidswith Fmoc-D-aminoacid derivatives. The retro-inverso peptide, i.e., theD CD28 peptide has the sequence EIGNSRENDLYPPPYMFIEK, SEQ ID NO. 6

[0058] As control, all D-amino acid CD28 enantiomers, and a CD28 peptidewith the amino acid sequence in the reverse order were also synthesized.The CD 28 peptide mimetics and the control peptides are summarized inTable 1 below. TABLE 1 Amino acid sequences of CD28 peptide mimetics andcontrol peptides CD28 Peptide Sequence Abbreviation Identity NH₂KIEFMYPPPYLDNERSNGTICOOH L-CD28 Free L peptideCH₃COL[KIEFMYPPPYLDNERSNGTI]LCONH₂ EL-CD28 End-blocked L-peptideCH₃COD[ITGNSRENDLYPPPYMFEIK]DCONH₂ RI CD28 Retro-inverso D-peptideCH₃COD[KIEFMYPPPYLDNERSNGTI]DCONH₂ D-CD28 D-peptide (Control)CH₃COL[ITGNSRENDLYPPPYMFEIK]LCONH₂ RL-CD28 Reverse-L-peptide (Control)

[0059] Peptide Synthesis and Purification:

[0060] The CD 28 peptide mimetics and control petides shown in Table 1above were synthesized by solid phase peptide synthesis followingFmoc/DCC/HOBt methodology on a fully automated peptide synthesizer(Model 396-5000 Multiple Peptide Synthesizer, Advanced Chemtech,Louisville, Ky.). The free L CD28 peptide was assembled on4-methylbenzhydrylamine resin (0.5 mmol/g substitution) with4-(hydroxymethyl) phenoxyacetic acid as the linker. The end groupblocked peptides were assembled on Fmoc-2,4-dimethyloxy-4′-(carboxymethyloxy)-benzylhydrylamine (Rink amide) resin(Advanced Chemtech, Louisville, Ky.) as peptide amides. Couplingreactions utilized six equivalents of each amino acid with1-hydroxybenzotriazole (HOBT),2-(1H-benzotriazole-1-yl)-1,1,3,3,-tetramethyluronium tetrafluoroborateand distilled diisopropylethylaime (DIEA). Deprotection was accomplishedwith 30% piperidine in DMF. Immediately after the final deprotectionstep, the free NH₂ group of the terminal amino acid residue wasacetylated with 5 ml of 3 mmol acetaimidazole in DMF (50 ml). Thecompletion of acetylation was confirmed by a negative Kaiser Ninhydrintest. With respect to the parent peptide, the retro-inverso peptide wasassembled in reverse order of amino acids with Fmoc-D-amino acidderivatives. The peptides were cleaved from the resin support withsimultaneous side-chain deprotection by acidolysis using TFA with 5%phenol, 5% thioanisole and 2.5% ethanedithiol as scavengers. The crudepeptides were purified by semi-preparative reverse phase highperformance liquid chromatography (RP-HPLC) using a C₁₈ column (10 mm by25 cm) (Vydac, Hesperia, Calif.) at a temperature of 32.5° C. and a flowrate of 5 ml/min. Peptides (5-10 mg per run) were loaded in 0.1M aceticacid and chromatographed for 30 min with a linear gradient of 10-60% ofacetonitrile in water containing 0.1% trifluoroacetic acid (TFA). Theseparations were monitored at 230 and 280 nm.

[0061] Analytical HPLC was run using a VIDAC C₁₈ column (4.6 mm by 25cm) using the same gradient as stated above. Eluants were monitored at214 and 254 nm. Purified peptideswere obtained in greater than 95%purity as assessed by reverse-phase HPLC. The identity of peptides wasfinally confirmed by matrix-assisted laser desorption/ionization time offlight mass spectrometry.

[0062] A. Sturctural Characterization of the Synthetic CD28 PeptideMimetics and Control Peptides

[0063] Circular dichroism (CD) spectroscopy is sensitive to thesecondary structure of globular proteins. CD spectroscopy is a usefultool for determining whether a polypeptide or protein adopts apolyproline (PP) II type helical conformation in solution.

[0064] To determine the nature of the secondary structure of thepeptides shown in Table I, CD spectra of CD28 peptide mimetics, as wellas the control peptides, were recorded at 25° C. Circular dichroismmeasurements were recorded at room temperature on an AVIV Model 62A DSspectrometer equipped with a thermostatic temperature controller andmicrocomputer as described previously Lairmore, M. D., A. M. DiGeorge,S. F. Conrad, A. V. Trevino, R. B. Lal, and P. T. Kaumaya. 1995. HumanT-lymphotropic virus type 1 peptides in chimeric and multivalentconstructs with promiscuous T-cell epitopes enhance immunogenicity andovercome genetic restriction. J Virol 69:6077). CD spectra were recordedin a quartz cell of 0.1 cm pathlength. Each spectrum was obtained byaveraging one nm/sec in the 190 to 270 nm wavelength range, using abandwidth of 1.0 nm and a response time of 1 s.

[0065] CD28 peptides were dissolved in PBS, pH7.4, in 50%trifluoroethanol (TFE) or in 4M −6 M CaCl₂ at 412 μM concentration forCD measurements. The CD spectra were recorded at a range of temperaturesbetween 5° C. and 90° C. Raw CD signals (in millidegrees) were convertedto mean residue ellipticity (MRW) in deg.cm²/dmol using the formula [θ]_(MRW)=[θ] _(obs)/101 cn where θ_(obs) is the observed ellipticity, 1 isthe path-length in cm, c is the molar concentration of peptide and N isthe number of residues in the peptide.

[0066] The CD spectrum of free L CD28 peptide mimetic showed a largeminimum at 202 nm (θ=−62.73×10³ deg.cm².dmol⁻¹) and a slight maximum at221 nm (θ=−3.5×10³ deg cm²dmol⁻¹)(data not shown) at 25° C. The CDspectrum of EL CD28 peptide mimetic presented a strong mean residuemolar ellipticity minimum at 205 nm (θ=−69×10³ deg.cm².dmol⁻¹) and aweak maximum at 221 nm (θ=−17×10³ deg cm².dmol⁻¹) although still in thenegative ellipticity region at 25° C. Compared to the free L CD28spectrum, the intensity of ellipticity near 200 nm was considerablyenhanced in the EL CD28 peptide mimetic, suggesting stabilization of thehelical secondary structure (data not shown). When the temperature wasraised to 90° C., the mean residue molar ellipticity was decreased(θ=−62.32×10³ deg cm².dmol⁻¹)(FIG. 1B). Similar variation withincreasing temperature has been observed in proline-rich peptides, dueto a transition from the PP II helical structure to a disordered randomcoil conformation (Antonyraj, K. J., T. Karunakaran, and P. A. Raj.1998. Bactericidal activity and poly-L-proline II conformation of thetandem repeat sequence of human salivary mucin glycoprotein (MG2). ArchBiochem Biophys 356:197). When the CD spectrum of EL CD28 peptidedissolved in 6M Ca Cl₂ was recorded at 25° C., the intensity of meanresidue ellipticity minimum at 205 nM was drastically decreased(θ=−28×10³ deg cm².dmol⁻¹) suggesting complete disruption of the helicalstructure (FIG. 1A). These observations are consistent with the CDspectrum of polypeptide sequences reported to prefer a PP II typehelical structure. (Sreerama, N., and R. W. Woody. 1994. Poly(pro)IIhelices in globular proteins: identification and circular dichroicanalysis [published erratum appears in Biochemistry May 30,1995;34(21):7288]. Biochemistry 33:10022.)

[0067] The CD spectrum of the RI CD28 peptide mimetic at 25° C.presented a mean residue ellipticity maximum at 205 nm (θ=57×10³ degcm².dmol⁻¹), a weak minimum at 215 nm (θ=1.14×10⁴ deg cm².dmol⁻¹) and aweak maximum at 223 nm (θ=18.3×10³ deg cm².dmol⁻¹) (FIG. 1A). Similarmirror-image like CD spectra of retro-inverso isomers of L peptides havebeen previously reported (Petit, M. C., N. Benkirane, G. Guichard, A. P.Du, M. Marraud, M. T. Cung, J. P. Briand, and S. Muller. 1999. Solutionstructure of a retro-inverso peptide analogue mimicking thefoot-and-mouth disease virus major antigenic site. Structural basis forits antigenic cross-reactivity with the parent peptide. J. Biol Chem274:3686.) By increasing the temperature to 90° C., the molarellipticity maximum markedly decreased in intensity (θ=17.2.9×10³ degcm².dmol⁻¹) and shifted to a longer wavelength (211 nM). The CD spectrumof the retro-inverso CD28 peptide mimetic dissolved in 6M CaCl₂ showed adramatic decrease in molar ellipticity maximum at 205 nM (θ=1.10×10⁴ degcm².dmol⁻¹), complete loss of molar ellipticity minimumat 215 nM and themaximum at 223 nM (FIG. 1B). These observations reflect thedestabilizingeffect of CaCl₂ on the PPII helical conformation adopted bythe retro-inverso CD28 peptide mimetic.

[0068] The CD spectrum of a six residue free peptide comprising the“MYPPPY” motif alone showed a weak minimum (θ=−8.6×10¹ deg cm².dmol⁻¹)at 208 nm (data not shown). This suggests that the length of the CD28peptide and the side chain interactions with the flanking residues playa role in the formation of a PP II helix.

[0069] B. Interaction of the CD 28 Peptide Mimetics with B7-1

[0070] Binding experiments were performed by surface plasmon resonance(SPR) on a BIAcore instrument from Pharmacia Biosensor (Uppasala,Sweden). All experiments were performed at 37° C. using HBS-EP buffer(25 mM Hepes, pH7.4, 150 nM NaCl, 3.4 mM EDTA and, 005% surfactant P20)supplied by Pharmacia Biosensor.

[0071] CD80-Ig (extracellular domain of CD80 fused with the constantregion of mouse IgG1 heavy chain) at 35 μg/ml in 10 mM sodium acetatebuffer, pH 4.2, was coupled to a research grade CM 5 sensor chip using astandard amine coupling procedure with the following modifications (38).To reduce the immobilization level of the ligand, the surface wasactivated for 3 (instead of 7) minutes with N-hydroxysuccinamide andN-hydroxymethyl-N-(3-diethylaminopropyl) carbodiimide. This typicallyresulted in immobilization of 1422-1660 RU of CD80-Ig on the sensorchip. Following coupling, noncovalently bound ligand was removed bywashing twice with 5 mM NaOH.

[0072] Kinetic analysis was performed by injecting the analytes (L-CD28,EL-CD28, RI CD 28, RL CD28 and D CD28 peptides) at 0.375 μM to 18.5 μMconcentrations in HBSS-EP, pH7.4, for 300 s with a flow rate of 10μl/min. The analytes were also injected at the same concentration andinjection times over an empty flow cell with nothing immobilized.

[0073] Competitive Kinetic Analysis.

[0074] Competitive kinetic analysis was performed as describedpreviously using purified anti-mouse IgG1 Fc (31437zz) (Pierce City,Ill., USA) to indirectly immobilize CD80-Ig (39). This resulted in286-342 RU of CD80-Ig bound to the chip. The binding kinetics of CD28-Igwas assessed by injecting a range of CD28-Ig concentration (187 nM to 12μM) over immobilized CD80-Ig (324 RU) for 5 min at 10 μl/min. CD80-Igwas regenerated using a 3 min injection of 5 mM NaOH. For competitivekinetic analysis, 100 μl of a 3.2 μM CD28-Ig solution was mixed with 100μl of EL CD28 or RI CD28 peptides (6.18 μM to 503 μM in HBS-EP) andinjected over the surface of CD80-Ig as secondary analyte for 5 min witha flow rate of 10 μl/min. The CD80-Ig surface was regenerated betweeninjections by washing for 3 min with 5 mM NaOH. The mixtures were alsoinjected on an empty flow cell with no protein immobilized, as acontrol.

[0075] Data analysis was performed with BIAevaluation software version2.1(Pharmacia Biosensor AB). The binding as measured in response units(RU) in BIAcore and the binding rate, dR/dt, can be used to evaluate thekinetics of the synthetic CD28 peptides-CD80-Ig interaction. Prior tokinetic analysis, data were adjusted to zero baseline level bysubtracting the background responses obtained by injection of theanalytes through a control flow cell with no ligand immobilized. Datafrom direct kinetic analysis were analyzed as follows. First, thedissociation rate constant, k_(d), (units: s⁻¹) was determined, byfitting experimental data from the buffer flow part of the sensogram tothe equation

R(t)=R ₁ *e ^(−kd*(t) ^(₀) ^(−t) ^(₁) ⁾  (1)

[0076] where t₀ is the injection time, R₁ is the response level at thestart of dissociation time t₁ and k_(d) is the dissociation rateconstant.

[0077] The k_(d) value obtained was used as a constant during theanalysis of the injection phase data. Binding data above the noise levelwas selected by converting the sensogramto a plot of the logarithm ofthe binding rate vs time (dR/dt vs. time). The k_(a) was determined bynonlinear curve fitting of the following equation

R(t)=R _(eq)*{1−e ^(−(k) ^(_(a)) ^(*C+k) ^(_(d)) ^()*(t))  (2)

[0078] where R(t) is the response at time t, R_(eq) is the steady stateresponse level, k_(a) is the association rate constant (units: M⁻¹s⁻¹),k_(d) is the dissociation rate constant and C is the concentration ofthe injected peptide analyte. An offset was added to account for therefractive index differences between the analyte and the running buffer.

[0079] In competitive kinetic experiments, the observed response R isthe sum of the contributions of R₁ and R₂ from the two analytes. Thebinding data was analyzed using the equation

R(t)=R _(1 (CD28-Ig)) +R _(2 (CD28-P))  (3)

where

R ₁ =R _(max) k _(a1) C ₁ /k _(f) −k _(s) {k ₁(k _(f) −k _(s))/k _(f) *k_(s) +k ₁ −k _(f) /k _(f) *e ^(−k) f ^((t−t0)) −k ₁ k _(s) /k _(s) *e^(−ks(t−t0))}

R ₂ =R _(max) MW ₁ k _(a2) C ₂ /MW ₂ *k _(f) −k _(s) [{k ₂(k _(f) −k_(s))}/k _(f) *k _(s)}+(k ₂ −k _(f) /k _(f))*e ^(−k) f ^((t−t0))−(k ₂ −k_(s) /k _(s))*e ^(−ks(t−t0))

k _(f)=0.5{k _(tq) +k ₂+π(k _(a1) −k ₂)²+4kf ₁ k _(CD28P) C ₁ C ₂}

k _(s)=0.5{k _(t1) +k ₂−π(k _(t1) −k ₂)²+4k ₁ k ₂ C ₁ C ₂}

k _(t CD28-Ig) =k _(a1) C+k _(d1)

k _(t2) =k _(a2) C ₂ +k _(d2)

[0080] When competitive data were analyzed, k_(a), k_(d) and R_(max)values obtained for the interaction between CD80-Ig and CD28-Ig wereused as non floating parameters and the rate constants for CD28 peptideswere calculated from the injection phase data using the expandedequation 3.

[0081] After analyzing sensograms obtained from injections of increasingconcentrations of peptide analytes or CD28-Ig, the experimental data wasanalyzed using the BIAsimulation software 2.1. A plot of the logarithmof (dR/dt) against time was calculated from the sensograms, and then theslopes at different concentrations were plotted against theconcentrations of the peptide analyte. Different angles obtained of thelinear lines represent the variation of affinities, which gives theassociation rate constant (k_(a)) from the equation

ln dR/dt=ln(k_(a)CRmax)−K_(a) C+k _(d))t  (4)

[0082] where dR/dt is the rate of change of the SPR signal, C is theconcentration of the analyte, R_(max) is the maximum analyte bindingcapacity in RU and R is the SPR signal in RU at time t. The dissociationrate constant is obtained from the equation

K _(d) =k _(d) /k _(a)  (5)

[0083] Sensograms from direct kinetic analyses of EL CD28 and RI CD28peptides mimetics at different concentrations from one experiment arerepresented in FIGS. 2 and 3 respectively. Both peptides reachedequilibrium binding very rapidly (12-20 s) and in the washing phasedissociated rapidly (10-12 s). Similar features of fast binding kineticshave been reported for the B7:CD28 interaction (33, 48). The backgroundresponses following injection of EL CD28 and RI CD28 peptide mimeticsover an empty flow cell with no protein immobilized are equivalent andvery similar to the responses obtained following injections of controlpeptides (data not shown). The maximum response units obtained forbinding to CD80-Ig was 478 RU, 667.5 RU and 444.2 RU for L CD28, EL CD28and RI CD28 peptide mimetics respectively. The response units observedat the beginning and end of the experiments were similar indicating thatthe bound CD80-Ig was stable. Dissociation of L CD28, EL CD28, and RICD28 peptides from bound CD80-Ig was analyzed from the buffer flow phaseof the sensogram and the dissociation rate constant (k_(d)) for eachcurve was determined using equation (1). The injection phase of thesensogram was analyzed using equation (2) by nonlinear curve fittingincorporating the calculated k_(d) value to obtain the k_(a) value foreach curve. The calculated parameter values with standard error arepresented in Table 2.

[0084] Dta for both k_(a) and k_(d) are quite consistent over the entirerange of concentrations used. The difference between experimental andcalculated data for both EL CD28 and RI CD28 peptide mimetics stimatedby Chi² value is low being close to the noise level of the instrument. Alinear regression plot of the rate of change in the response againstresponse units was plotted using these values of k_(a) and k_(d) foreach CD28 peptide analyte. The slope of this plot was then plottedagainst the concentration of the peptide to yield a K_(d) of 2.44 μM,2.34 μM and 2.53 μM for L CD28, EL CD28 and RI CD28 peptide mimetics,respectively, for binding to CD80-Ig. Consistent with the lack of PP IIhelix formation as observed by CD studies, a synthetic consisting of thehexapeptide motif alone did not bind CD80-Ig (data not shown). TABLE 2APR C (nM) k_(a) SE k_(a) Chi² k_(d) SE k_(d) Chi² EL CD28 6.00E+031.11E+05 7.96E+03 4.07E−01 2.73E−01 2.52E−02 1.22E+00 3.00E+03 1.37E+051.10E+04 7.02E−01 0.214 0.0283 9.05E−01 1.50E+03 1.15E+05 9.33E+032.20E−02 2.42E−01 9.33E−02 1.02E+00 7.50E+02 1.10E+05 1.23E+04 2.92E−022.74E−01 5.00E−02 1.23E+00 3.75E+02 2.46E+05 5.63E+04 2.04E−01 3.99E−012.49E−02 1.31E+00 RI CD28 6.00E+03 1.12E+05 9.05E+03 4.74E−04 2.28E−013.47E−02 6.75E−02 3.00E+03 1.22E+05 2.31E+04 3.96E−03 1.46E−01 1.67E−021.26E−02 1.50E+03 1.41E+05 5.14E+04 1.00E−01 1.83E−01 2.79E−02 7.12E−021.00E+03 1.71E+05 5.77E+04 1.12E−02 2.05E−01 3.09E−02 4.41E−02

[0085] In the method used for competitive kinetic analysis, indirectimmobilization resulted in 286-342RU of CD80-Ig bound via anti mouseIgG1Fc mAB coupled to the sensor chip surface. FIG. 4a and 4 b arerepresentative sensograms obtained from the competitive experimentsusing the CD28-Ig and ELCD28 and RICD28 APR respectively. The top curveis the interaction of CD28-Ig (3.2 μM) alone with the CD80-Ig. Theresponse level gradually goes down as the concentration of the specifiedCD28 peptide increases. The values obtained from the kinetic analysisfor the interaction of CD28-Ig (MW=50,264.36 Da, k_(a)=1.38×10⁵M⁻¹s⁻¹,k_(d)=0.563 s⁻¹) were used as invariant parameters in competitivekinetic analysis with EL CD28 and RI CD28 (MW=2425.25 Da) peptides andvalues for the association and dissociation rate for competing CD28peptide analytes were floated while fitting the injection phase of thecurves to the expanded equation (3). The calculated parameter values aregiven in Table3. The average dissociation constant for EL CD28 peptidemimetic binding to CD80-Ig (K_(d)=2.79 μM +/−1.32) compares well withthe value obtained with direct binding of the peptide to CD80-Ig(K_(d)=2.34 μM). However, the dissociation constant varied with varyingconcentration of EL CD28 peptide mimetic(Table 3).

[0086] The average value of dissociation constant for RI CD28 peptidemimietic binding to CD80-Ig (K_(d)=1.75 μM=/−0.67) is a little lowerthan the value obtained with direct TABLE 3 APR C (nM) k_(a) SE k_(a)k_(d) SE k_(d) Chi². K_(d) (μM) EL CD28    3 × 10⁵ 2.36 × 10⁶  9.29 ×105 6.89 2.66 1.35 2.92    2 × 10⁵ 5.51 × 10⁴  1.68 × 103 8.62E−022.78E−02 0.125 1.56    1 × 10⁵ 3.16 × 104 2.38 × 103 8.03E−02 1.15E−020.841 2.54  0.5 × 10⁵ 6.32 × 104  5.3 × 103 3.16E−01 2.31E−02 1.24 5 0.25 × 10⁵ 3.36 × 103  4.3 × 103 1.95E−02 5.65E−02 0.0947 3.36 0.125 ×10⁵ 7.34 × 104 4.61 × 103 1.01E−01 2.55E−02 1.89E+00 1.38 RI CD28Concentrat k_(a) SE k_(a) k_(d) SE k_(d) Chi². K_(d) (μM)    3 × 10⁵1.07E+04 8.30E+02 0.0206 0.0144 1.61 1.93    2 × 10⁵ 2.35E+04 1.76E+030.0245 0.0252 1.41 1.04    1 × 10⁵ 9.47E+04 5.72E+02 0.142 0.0112 0.3591.5  0.5 × 105 9.24E+04 2.32E+03 0.275 0.0252 0.0578 2.98  0.25 × 1055.44E+04 3.38E+03 0.0738 0.0114 0.685 1.36 0.125 × 105 1.05E+05 8.17E+030.177 0.0271 0.53 1.69

[0087] binding of the peptide to CD80-Ig (K_(d)=2.53 μM). The values ofdissociation constant obtained with varying concentrations of RI CD28peptide mimetices were more consistent (Table 3).

[0088] Synthetic CD28 peptides competed efficiently with CD28-Ig to bindB7-1. These results demonstrate that CD28 peptides form complexes withB7-1 and represent a ligand binding epitope.

Example 2 Inhibition of T cell Proliferation and Activation Using thePeptides of Example 1

[0089] The in vitro effect of varying concentration of the CD 28 peptidemimetics on CD4+ lymph node cells and spleen cells from transgenic micebearing the Vα4 Vβ8.2 TCR specific for MBP Ac 1-11 was determined.

[0090] Antigens: MBP was extracted from guinea pig (GP) spinal cords(Harlan Sprague Dawley, Indianapolis, Ind.). MBP NAc1-11 peptide wassynthesized.

[0091] Proliferation Analysis:

[0092] CD4+ T cells were purified from pooled peripheral lymph nodes(inguinal, axillary, brachial, cervical, popliteal), and mesentericlymph nodes and spleen of 6-8 wk old Vα4/Vβ8.2 TCR transgenic mice bypositive selection. Single cell suspensions were prepared by lympholyteM gradient, washed, counted and incubated with magnetic bead conjugatedanti-mouse CD4 (L3T4) (Miltenyi Biotech Corporation) followed bypositive selection using magnetic selection columns. Purity of the CD4⁺cells was greater than 90% as assessed by staining with FITC labeledanti-mouseVβ8.2 mAb (Pharmingen). During the selection process, T cellsmaintain a naïve phenotype with no evidence for T cell activation asmeasured by proliferation in culture. Purified CD4+ T-cells (5×10⁴cells/well) were cultured together with wild type splenocytes fromB10.PL mice as APC's (10⁵ cells/well) in RPMI 1640 containing 10% FCS,25 mM HEPES, 2 mM L-glutamine, 50 U/ml penicillin, 50 μg/mlstreptomycin, and 5×10-5 M 2-ME in round-bottom 96-well plates and MBP(40 μg/ml) or MBP NAc1-11 (10 μg/ml) or media only in the presence orabsence of synthetic CD28 peptide analogues at different concentrationsin triplicate wells for 72 h, including a final 18-h pulse with [³H]thymidine. Cultures were harvested onto glass-fiber mats using a Skatronharvester (Skatron, Sterling, Va.) and were counted by liquidscintillation on LKB Betaplate (LKB, Rockville, Md.). The means of thetriplicate were determined and the results are expressed as delta cpm(mean counts per minute of cultures with Ag-mean counts per minute ofcultures with medium alone)±SD.

[0093]FIG. 5 shows a significant decrease in the proliferative responsesof CD4+ LNC and spleen cells to MBP Ac1-11 when treated with EL CD28 orRI CD28 peptide mimetics, and the effect is not dose dependent. Maximuminhibition was observed in CD4+ LNC at 120 μM concentrations of L CD28(59.5%) followed by EL CD28 (47.6%) and RICD28 (45.7%) peptide mimetics.The proliferative responses of CD4⁺ splenocytes were also decreased butto a lesser extent with the observed maximum inhibition of 50.2%, 38.2%and 42% with L CD28, EL CD28 and RI CD28 peptide mimetics respectively.A similar decrease in the proliferative responses to MBP was alsoobserved (data not shown).

[0094] The control RL CD28 and D CD28 peptides did not show inhibition.The hexapeptide consisting of the hydrophobic motif only did not showinhibition of T cell-proliferation. The hexapeptide consisting of thehydrophobic motif only did not show inhibition of T cell-proliferation.These results demonstrate that treatment with synthetic CD 28 peptideeffectively blocked the expansion of encephalitogenic T cells in vitrosuggesting the feasibility of a therapeutic application for thesepeptides in vivo.

[0095] Augmentation of T cell proliferation following CD28 ligation is aresult of its ability to increase the synthesis of IL-2. To determinewhether the observed reduction in proliferation is reflected in the IL-2secretion, the ELISPOT assay was performed.

[0096] Ninety-six well unifilter plates (Polyfiltronics, Rockland, Md.)were coated overnight at 4° C. with rat anti-mouse IL-2 (CloneJES6-1A12) (Pharmingen, Calif.) at 4 μg/ml. The plates were then washed4× with sterile PBS and blocked with 1% BSA in DMEM for 2 h at roomtemperature. Subsequently, isolated single cell suspensions of CD4+ LNC(5×10⁶ cells/ml) and splenocytes (10×10⁶ cells/ml) were added with theantigens and CD28 peptides under the conditions as specified for theproliferation assays. After 24 h of culture in the incubator, the cellswere removed by washing 3× with PBS and 4× with PBS containing Tween(1:2000) (PBST). Then biotinylated anti mouse-IL-2 (Clone JES6-5H4)(Pharmingen, Calif.) 2 μg/ml was added and incubated at 4° C. overnight.After washing 3× with PBST and 3× with PBS, goat anti-biotin antibodyconjugated to alkaline phosphatase (Vector Laboratories Inc, Burlingame,Calif.) diluted to 1:1000 in PBST containing 1% BSA was added andincubated for 2 h at room temperature. The spots were visualized byadding BCIP/NBT phosphatase substrate (Kirkeguard & Perry Laboratories,Gaithesburg, Md.). Image analysis of ELISA spot assays was performed ona Series 1 Immunospot Image Analyzer (Autoimmun Diagnostika (US) Inc.)customized for analyzing ELISA spots to meet objective criteria forsize, chromatic density, shape and color.

[0097]FIG. 6 shows that MBP stimulated CD4+ LNC bearing Vα4 Vβ 8.2 TCRexhibited significantly reduced frequency of IL-2 secreting cells in thepresence of 50 mM EL CD28 or RI CD28 peptide mimetics when compared tountreated cells. There was an increase in the frequency of IL-2secreting antigen stimulated CD4+ LNC when higher concentrations of CD28peptides were used. This observation perhaps reflects induction ofapoptosis, since IL-2 is also known to sensitize activated T cells tocell death (50).

[0098] Detection of Apoptosis:

[0099] To investigate whether the observed decrease in proliferativeresponses following treatment with CD28 peptides is a result of cellloss, cell death was measured by enzymatic in-situ labeling. Apoptoticcells among MBP NAc1-11 stimulated TCR Vβ 8.2+ CD4+ T cells weredetected by staining with FITC labeled Tdt (terminal deoxynucleotidyltransferase). Cells incubated in DNAse served as positive control.

[0100] CD4+ T cells from pooled lymph nodes and spleen from B10.PL TCRVβ 8.2 Vα 4 transgenic mice were cultured in RPMI 1640 in a 96 wellround-bottom plate, essentially under the conditions specified forproliferation assays. Cells were harvested at the end of 48 hours andapoptotic cells among TCR Vβ 8.2+ CD4+ T cells were detected by theTUNEL method. Briefly, after washing twice with PBS containing 1-% ratserum, the cells were stained with PE labeled anti-mouseVβ 8.1,8.2 TCR(clone MR5-2) (Pharmingen, San Diego, Calif.) for 30 min at roomtemperature and washed. Subsequently, the cells were fixed with 4%paraformaldehyde in PBS for 30 min at room temperature. Then the cellswere washed and permeabilized with 0.1% Triton X-100 in 0.1% sodiumcitrate for 2 min at 4° C. After washing, the DNA strand breaks in thecells were detected by incubating the cells with fluorescein labeledTUNEL mix (terminal deoxynucleotidyl transferase (Tdt)) for 60 min at37° C. using an in situ cell death detection kit (Boehringer Mannheim,Mannheim, Germany) according to the manufacturer's recommendations. As apositive control, cells were incubated in DNAse (1 mg/ml) for 10 minutesat room temperature prior to incubation with TdT. The negative controlconsisted of cells incubated without TdT. Cells were then washed withPBS and analyzed by flow cytometry.

[0101] Treatment with 150 μM ELCD28 or RI CD28 peptides at the time ofantigen stimulation resulted in a significantly higher percentage ofantigen specific CD4+ LNC undergoing apoptosis after 48 hr culture whencompared to untreated cells (15.4%) (FIG. 7). CD4⁺Vβ8.2⁺ spleen cellsalso exhibited increase in apoptosis when treated with 150 μM ELCD28 orRI CD28 peptide (data not shown).

[0102]FIG. 7a is a representative histogram of three differentexperiments showing increased apoptosis of the CD4+ LNC cultured in thepresence of indicated CD28 APR. Control cells cultured in the absence ofantigen were 80% apoptotic (data not shown). These results suggest thatthe synthetic CD28 effectively competes with cell surface CD28 forbinding B7-ligands on the APC and blocks the costimulation required forsustained survival of antigen stimulated T-cells.

[0103] The functional efficacy in vitro, suggests a therapeuticpotential for the CD28 peptide mimetics in various disease conditionsrequiring downregulation of T cell responses such as autoimmunediseases, certain chronic infections and graft-versus host disease.

Example 3 Treatment of EAE with the CD 28 Peptide Mimetics of Example 1

[0104] Most studies on costimulatory blockade in EAE have focused on thesuppression of T cell responses during antigen priming and diseaseinduction. In this Example, the ability of CD28 peptide mimetics tosuppress ongoing clinical disease in EAE animals is demonstrated.

[0105] Mice:

[0106] Female B10.PL mice (6-8 wk old) were obtained from The Jacksonlaboratory (Bar Harbor, Me.) and housed at the Ohio State University,(Columbus, Ohio).

[0107] Induction of EAE and CD28 Peptide Treatment:

[0108] Mice were injected subcutaneously over four sites on the flankwith an emulsion containing 200 μg of guinea-pig-MBP in CFA containing200 μg heat killed Mycobacterium tuberculosis, Jamaica strain. Pertussistoxin (List Biological, Campbell, Calif.) at 150 ng in 0.2 ml of PBS wasgiven intraperitoneally at the time of immunization and 48 hours later.Animals were observed daily for clinical signs and scored as follows:

[0109] 1, limp tail or waddling gait with tail tonicity; 2, waddlinggait with limp tail (ataxia); 2.5 ataxia with partial paralysis of onelimb, 3, partial hind-limb paralysis, 3.5 full paralysis of one limbwith partial paralysis of the second limb, 4, full paralysis of twolimbs, 4.5 moribund and 5, death. All mice immunized with MBP (200 μg)in CFA developed clinical disease varying between ataxia and completehind limb paralysis by day 14 post-immunization. They were thendistributed randomly into six groups with mean clinical score of eachgroup approximately the same.

[0110] Groups of mice were left untreated or injected intravenously with500 μg of EL CD28 or RI CD28 peptide mimetics, or LCD28, RLCD28 andDCD28 peptides. The mean clinical score of untreated mice continued toincrease reaching a maximum of 3.4 on day 20. Similarly the diseasecontinued to progress in mice treated with LCD28, RLCD28 and DCD28,reaching mean maximal clinical score of 3.8 (day 18), 3.4 (day 16 ) and3.7 (day 16) respectively. In contrast mice treated with EL CD28 and RICD28 peptide mimetics continued to show clinical improvement from day 16throughout period of observation (26 days) post-immunization.(FIG. 8Aand B). This indicates that short-term blockade of CD28 costimulationwas capable of attenuating ongoing disease progression in EAE.

[0111] The biological activity of synthetic CD28 peptide analoguesduring antigen priming in vivo in EAE was then shown. B10.PL miceimmunized with GP-MBP (200 μg) in CFA were either left untreated orinjected intravenously 500 μg of EL CD28 or RI CD28 peptide mimetics orLCD28, RLCD28 and DCD28 peptides on the day of immunization. The vehicletreated mice and mice treated with: LCD28, RL CD28 and D CD28 peptidehad maximum disease incidence of 100%, 100%, 91.5% and 91.5% and maximummean cumulative score per day of 1.9, 1.8, 1.7 and 2 respectively. Incontrast, significant inhibition of EAE was observed with mean maximumincidence of 70% and 60% and a maximum mean cumulative score of 1.1 and0.74 in mice treated with EL CD28 and RI CD28 peptide mimectics,respectively. The effect of RI CD28 peptide injection lasted for theduration of observation (37 days) in one experiment.

[0112] The data also show that single administration of synthetic CD28peptide analogs ameliorated ongoing EAE in B10.PL mice. Groups of miceimmunized with MBP in CFA were left untreated or injected intravenously500 μg of EL CD28 or RI CD28 or control CD28 peptides on day 14, whenmost mice exhibited EAE with a minimal score of 2.0 (FIG. 8B)

Example 4 Treatment with Synthetic CD28 Peptide Mimetics Decreased IL-2Production by Encephalitogenic T cells In Vivo.

[0113] MBP reactive T cells that induce EAE are known to display a Th₁phenotype secreting the proinflammaotry cytokines IL-2, INF-γ and TNF-β(36. Kay, B. K., M. P. Williamson, and M. Sudol. 2000. Faseb J 14, no.2:231). ELISPOT assay was used to assess in vitro cytokine production bydraining lymph node cells and splenocytes upon restimulation from invivo MBP-primed T cells. Mice were treated as described in Example 3, onday 0 of immunization. The frequency of IL-2 secreting lymph node cellsdecreased significantly EL-CD28 (2776+/−53.5; p<0.05) and RI CD28(1753+/−37.7; p<0.01) treated mice as compared to vehicle (4846+/−14.3)and control RL-CD28 (3386+/−23.3) or D CD28 (2675+/−14.2) peptidetreated mice.

Example 5 CD28 Peptide Mimetics Protect from EAE by Inducing Apoptosisof CD4+ T Cells In Vivo.

[0114] This study showed that the protective effects of CD28 peptidemimetics in EAE were mediated through apoptosis and quantified theeffects. This was done by quantifying the DNA strand breaks in CD4+lymphocytes detected by enzymatic labeling of nicked ends. Cellsincubated in DNAse served as positive control. A significantly higherpercentage of CD4+ T cells were apoptotic in mice treated with RI CD28peptide mimetics (16%) on the day of immunization as compared to vehicletreated mice (8.7%).

What is claimed is:
 1. A CD28 peptide mimetic for blocking deleterious Tcell mediated immune reaction, said peptide mimetic being from 15 to 30amino acids in length, said peptide mimetic comprising levoratory ordexorotary amino acids, said peptide mimetic comprising a core motifdispersed between two flanking sequences, each of said flankingsequences comprising a plurality of amino acids; wherein the sequence ofthe core motif is MYPPPY, SEQ ID NO. 1, when the peptide mimeticcomprises levoratory acids; wherein the sequence of the core motif isYPPPYM, SEQ ID NO. 2, when the peptide mimetic comprises dexoratoryacids; and wherein the peptide mimetic assumes a polyprolineconformation when placed in water at physiological pH and a temperatureof about 25° C.
 2. The peptide mimetic of claim 1 wherein the flankingsequences are amphiphilic, anti-parallel, right-twisted B strands andcharged amino acid residues.
 3. The peptide mimetic of claim 1 whereinthe amino and carboxyl ends of the peptide are end blocked.
 4. Thepeptide mimetic of claim 1 wherein the peptide mimetic binds to the B71ligand with an affinity that is from 10 fold greater to 2 fold less thanCD
 28. 5. The peptide mimetic of claim 1 wherein the affinity of thepeptide mimetic for the B71 ligand is less than the affinity of CTLA-4for the B71 ligand.
 6. The peptide mimetic of claim 1 wherein the Kd ofthe mimetic with respect to B71 is from 2 to 3 micromoles.
 7. Thepeptide mimetic of claim 1 wherein the flanking sequences of the L formof the peptide mimetic comprises a repetitive LS sequence, and whereinthe flanking sequence of the D form of the peptide mimetic comprises arepetitive SL sequence.
 8. The peptide mimetic of claim 1 wherein saidpeptide mimetic comprises the sequence of SEQ ID NO.
 3. 9. The peptidemimetic of claim 1 wherein said peptide mimetic comprises the sequenceof SEQ ID NO.
 4. 10. The peptide mimetic of claim 1 wherein said peptidemimetic comprises SEQ ID NO. 5 or SEQ ID NO.
 6. 11. The peptide mimeticof claim 1 wherein said peptide mimetic comprises SEQ ID NO. 7 or SEQ IDNO.
 8. 12. The peptide mimetic of claim 1 wherein said peptide mimeticcomprises SEQ ID NO. 9 or SEQ ID NO.
 10. 13. The peptide mimetic ofclaim 1 wherein said peptide mimetic is a biologically active variant ofa peptide mimetic which comprises one of the following referencesequences: SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQID NO. 9 or SEQ ID NO. 10; and wherein the sequences which flank thecore motif of the variant are at least 70% identical with the sequenceswhich flank the core motif in one of said reference sequences.
 14. Thepeptide mimetic of claim 13 wherein the sequence which flank the coremotif of the variant are at least 80% identical with the sequences whichflank the core motif in one of said reference sequences.
 15. The peptidemimetic of claim 13 wherein the sequence which flank the core motif ofthe variant are at least 90% identical with the sequences which flankthe core motif in one of said reference sequences.
 16. The peptidemimetic of claim 13 wherein said peptide is from 17 to 25 amino acids inlength.
 17. A method of treating a subject with a T cell mediateddisorder or autoimmune disease comprising: administering a biologicallyeffective amount of one or more CD 28 peptide mimetics of claim 1 tosaid subject.
 18. The method of claim 17 wherein the subject hasmultiple sclerosis, rheumatoid arthritis, insulin-dependent diabetesmellitus, or has received or is about to receive an allografttransplant.
 19. The method of claim 17 wherein the sequences which flankthe core motif of the peptide mimetic are amphiphilic, anti-parallel,right-twisted B strands and charged amino acid residues.
 20. The methodof claim 17 wherein the amino and carboxyl ends of the peptide are endblocked.
 21. The method of claim 17 wherein the peptide mimetic binds tothe B71 ligand with an affinity that is from 10 fold greater to 2 foldless than CD
 28. 22. The method of claim 17 wherein the affinity of thepeptide mimetic for the B71 ligand is less than the affinity of CTLA-4for the B71 ligand.
 23. The method of claim 17 wherein the Kd of themimetic with respect to B71 is from 2 to 3 micromoles.
 24. The method ofclaim 17 wherein said peptide mimetic comprises the sequence of SEQ IDNO. 3, or the retro inverso isomer thereof.
 25. The method of claim 17wherein said peptide mimetic comprises the sequence of SEQ ID NO.
 4. 26.The method of claim 17 wherein said peptide mimetic comprises SEQ ID NO.5 or SEQ ID NO.
 6. 27. The method of claim 17 wherein said peptidemimetic comprises SEQ ID NO. 7 or SEQ ID NO.
 8. 28. The method of claim17 wherein said peptide mimetic comprises SEQ ID NO. 9 or SEQ ID NO. 10.29. The method of claim 17 wherein said peptide mimetic is abiologically active variant of a peptide mimetic which comprises one ofthe following reference sequences: SEQ ID NO. 5, SEQ ID NO. 6, SEQ IDNO. 7, SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO. 10; and wherein thesequences which flank the core motif of the variant are at least 70%identical with the sequences which flank the core motif in one of saidreference sequences.
 30. The method of claim 17 wherein said peptidemimetic is from 17 to 25 amino acids in length.
 31. The method of claim1 wherein the flanking sequences of the L form of the peptide mimeticcomprises a repetitive LS sequence, and wherein the flanking sequence ofthe D form of the peptide mimetic comprises a repetitive SL sequence.32. A method of blocking activation and proliferation of CD4+ cellscomprising contacting such cells with oneor more CD 28 peptide mimeticsof claim 1.